In article <D5FG0n.1qM at demon.co.uk>, Anthony Tomlinson <tomlinson at pplros.demon.co.uk> writes:
>In article <3js36s$e27 at thot.u-strasbg.fr> Francois Nantel,
>Nantel at titus.u-strasbg.fr writes:
>>Dear bionetters,
>>>>I presently have big ligation problems but that was expected. I need to
>>put a 3kb insert into a 3.2 kb vector. Oh, did I tell you it is a blunt
>>ligation :-0
>>>>I tried treating the vector with alkaline phosphatase but the ligation
>>effeciency is so low I didn't get anything.
>>>>If anybody have a knock-out technique for hard to do ligation, I would be
>>very grateful.
>>There is no real special technique, you just have to accept that it
is not going to be as efficient as any sticky end-ligation. All you need
for success is: phosphatased, gel purified vector; a lot of ligase ( 1-5
Weiss units in 10 ul reaction mixture) and an excess of insert. Typically,
20 ng of vector and 20-100 ng of an insert are ligated in 10 ul. With
average competent cells ( 10E7 colonies per ug ) this reaction should produce
a total of 200 colonies, of which at least 30% are recombinant. These
figures in fact show that such a ligation is about 10E4 times less efficient
than an average sticky-end one. Hope this helps,
*************************************************************************
Alexander Kraev, Ph.D. Internet: bckraev at aeolus.ethz.ch
Lab. of Biochemistry III Phone: 0041-1-632-31-47
Swiss Federal Institute of Technology FAX: 0041-1-632-12-13
Universitaetstr.16
CH-8092 Zurich
"Some ideas are obscure not because they are complex, but because they
are excluded from our circle of comprehension" - Kozma Prutkov